Heat shock proteins (Hsps) are constitutively expressed in many tumors, suggesting that these proteins provide a selective advantage to cells in tumor development. Expression of recombinant Hsp72 enhances tumorigenicity, while abrogation of Hsp72 from tumor cells (but not normal cells) reduces their tumorigenic properties and sensitizes to certain drugs. The first goal of this proposal is to elucidate the role of the elevated expression of Hsp72 in cancer cells. We hypothesize that Hsp72 allows tumor cells to escape apoptosis induced by activated oncogenes and to survive toxic conditions of tumor microenvironments. This hypothesis will be evaluated with immortalized mammary epithelium cells that over-express c-myc oncogene, and the mechanisms of the Hsp72-mediated suppression of apoptosis will be elucidated. Experiments with transgenic mice that overexpress both c-myc and Hsp72 in mammary tissue will test the significance of these findings for cancer development. Specific depletion of Hsp72 in some tumor cell lines leads to enhanced sensitivity to certain anti-cancer agents, and to reduced growth rate and anchorage-independent growth, which coincide with downregulation of the NF-kappaB signaling pathway. We will test a hypothesis that downregulation of this pathway is responsible for enhanced drug sensitivity and reduced tumorigenic properties of cancer cells upon depletion of Hsp72. The second goal is to establish the role of the heat shock response in cancer cell resistance to proteasome and Hsp90 inhibitors. Hyperthermia, proteasome inhibitors (e.g. Velcade) and Hsp90 inhibitors (e.g. 17-AAG) are three novel anti-cancer treatments, which strongly induce heat shock proteins. We hypothesize that inhibition of the heat shock response could enhance the potency of these treatments. In fact, deletion of the Hsfl transcription factor in MEF cells leads to a block of Hsps induction after hyperthermia, Velcade and 17-AAG, and enhances the sensitivity to these treatments. Hsf1 will be abrogated by siRNA in multiple myeloma, prostate and breast tumor cell lines, and sensitivity of these clones to Velcade and 17-AAG will be tested. With xenograft models, we will establish if downregulation of Hsf1 enhances tumor sensitivity to Velcade and 17-AAG. We have screened 35,000 compounds from several chemical libraries and identified 9 molecules, which potently (in micromolar and sub-micromolar concentrations) inhibit induction of Hsps by heat shock, Velcade and 17-AAG. We will analyze analogs of the two most promising compounds to identify critical elements in their structures and obtain non-toxic potent specific inhibitors of induction of Hsps. These inhibitors will be tested for their ability to enhance the potency of Velcade and 17-AAG in tumor cell cultures. These experiments will test a hypothesis that inhibitors of the heat shock response can enhance the anti-cancer activities of proteasome and Hsp90 inhibitors.